The essence of this research is the development of human cell lines and methods suitable for the routine production of human hybridomas, comparable to those established for successfully utilized murine systems. The selection of a variant of the human lymphoblastoid cell line WI-L2-729-HF[unreadable]2[unreadable] which does not produce immunoglobulin (Ig) and retains a high fusion efficiency is our first goal. To date clones that produce no detectable IgM or IgG have been obtained. However, since these clones continue to produce kappa chains, albeit at reduced levels, in the coming year these will be further selected upon to obtain a variant negative for both heavy and light chains. Variants will be analyzed as to the nature of the defect, for example, DNA mutation or deletion, or mRNA production. Most importantly, Ig-negative variants will be tested to determine their fusion efficiency in hybridoma formation, and also to ascertain that endogenous WI-L2-729-HF[unreadable]2[unreadable] Ig is not synthesized as a result of fusion. Our next goal is to define optimal conditions for generating specific hybridomas from human B-cells. In particular, in vitro immunization procedures and the role of growth and maturation factors in inducing antigen-specific B-cells to a stage optimal for fusion with WI-L2-729-HF[unreadable]2[unreadable] will be investigated. Experiments will continue to focus on sheep red blood cells (SRBC) as antigen, in which resulting hybridomas will be tested for anti-SRBC production by a plaque assay. Fusions involving soluble protein antigens, such as tetanus toxin, will be assayed by a solid-phase binding assay (ELISA) of secreted antibodies. The large-scale growth of human hybridomas should provide human monoclonal antibodies in high yield for future applications, including possible clinical utilization. In the coming year, growth in vitro (in serum-free medium) and in immune-deficient mice pretreated with pristane will be investigated in greater detail in order to maximize yield and to achieve highly purified antibodies from either culture supernatants or ascites fluid from tumor-bearing mice. (2)